Method of producing submicron particles of a labile agent and use thereof

ABSTRACT

The present invention relates to a sustained release composition comprising micron particles of labile agent and a method of preparing and using the sustained release composition. The invention further relates to micron particles of a labile agent and a method of preparing the micron particles. 
     The method of the invention for preparing a composition for the sustained release of a labile agent, comprises forming a suspension comprising the labile agent dispersed in a polymer solution comprising at least one biocompatible polymer and at least one polymer solvent. The suspension is then wet milled to achieve micron particles of the labile agent. The polymer solvent is then removed resulting in a solid polymer/labile agent matrix. The composition for sustained release of a labile agent is likewise prepared according to the method of the invention. 
     The sustained release composition of the present invention can be used in a method for providing a therapeutically effective blood level of a labile agent, in a subject in need of treatment with said agent, for a sustained period comprising administering to the subject the sustained release composition described herein. 
     The method for preparing micron particles of a labile agent comprises forming a suspension comprising the labile agent, dispersed in a polymer solution comprising at least one biocompatible polymer and at least one polymer solvent, and wet milling of the suspension. The submicron particles of labile agent, as described herein, are prepared according to this method.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.09/321,091, filed May 28, 1999, now U.S. Pat. No. 6,444,223. The entireteachings of the above application are incorporated herein by reference.

BACKGROUND OF THE INVENTION

It is known in the pharmaceutical industry that the rate of dissolutionof a particulate drug can increase with increasing surface area (e.g.,by decreasing particle size). This increase can result in enhancedbioavailability of the particulate drug. In sustained releasecompositions where a drug particle is dispersed within a matrix, forexample, a polymer matrix, improvements in release profiles aretypically seen as a result of a reduction in the particle size of thedispersed drug. Therefore, it is often desirable to minimize and controlthe particle size of a drug.

SUMMARY OF THE INVENTION

The present invention relates to a sustained release compositioncomprising micron particles of a labile agent and a method of preparingand administering the sustained release composition. The inventionfurther relates to micron particles of labile agent and a method ofpreparing the micron particles. The micron particles have a volumemedian particle size of less than about 2 microns. In a preferredembodiment, the particles are submicron particles having a volume medianparticle size of less than 1 micron.

The method of the invention for preparing a composition for thesustained release of a labile agent, comprises the steps of:

a) forming a suspension comprising the labile agent dispersed in apolymer solution comprising at least one biocompatible polymer and atleast one polymer solvent;

b) wet milling the suspension to achieve micron particles of the labileagent; and

c) removing the polymer solvent thereby forming a solid polymer/labileagent matrix.

The method can further comprise the step of forming droplets of themilled suspension prior to removal of the polymer solvent. Further, themethod can comprise freezing the droplets prior to removal of thepolymer solvent. According to the method of the invention, the dropletscan be microdroplets. In a specific embodiment wherein droplets areformed and then frozen, the polymer solvent can be removed by anevaporation and/or extraction process. In a preferred embodiment, themicron particles of labile agent are submicron in size.

The composition for sustained release of a labile agent is likewiseprepared according to the method of the invention as described above. Inother words, the composition for the sustained release of a labile agentas described herein is a composition prepared by the method comprisingthe steps of:

a) forming a suspension comprising the labile agent dispersed in apolymer solution comprising at least one biocompatible polymer and atleast one polymer solvent;

b) wet milling the suspension to achieve micron particles of the labileagent; and

c) removing the polymer solvent thereby forming a solid polymer/labileagent matrix.

The method for preparing micron particles of a labile agent comprisesthe step of (a) forming a suspension comprising the labile agent,dispersed in a polymer solution comprising at least one biocompatiblepolymer and at least one polymer solvent; and (b) wet milling thesuspension. In a preferred embodiment, the particles of labile agent aresubmicron particles.

The micron particles of labile agent, as described herein, are preparedaccording to the method of the invention. Consequently, the micronparticles of labile agent are prepared by forming a suspensioncomprising the labile agent, dispersed in a polymer solution comprisingat least one biocompatible polymer and at least one polymer solvent, andwet milling the suspension.

The method described herein as compared to other known methods ofparticle size reduction, provides micron particles of proteins, peptidesand oligonucleotides without agglomeration of the particles and withretention of biological activity. As shown in Example 1, known methodsof particle size reduction such as sonication, are capable of achievingparticles having a size of approximately 3 microns. However, achieving aparticle size of about 2 microns or less without degradation of thelabile agent can be readily achieved using the method described herein.

Importantly, the micron particles of labile agent once formed can,without isolation, be further processed to prepare a composition for thesustained release of labile agent. In addition, the sustained releasecompositions, which are prepared according to the claimed method,exhibit lower initial release of labile agent as a result of the micronparticle size which they possess, thereby providing increasedtherapeutic benefits resulting from low peak serum concentrations and/orlonger sustained duration.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the % release in vitro (% of theoretical load)and the C_(max) in vivo of human growth hormone (hGH) over the firsttwenty-four hours from the hGH-containing microparticles of Example 4,as a function of the particle size of the Zn⁺²-complexed hGH prior toencapsulation. Doses were normalized based on the weight of the testanimal.

FIG. 2 is a plot of the amount of hGH in serum (ng/mL) at predeterminedintervals over the first 48 hours following administration of thehGH-containing microparticles of Example 4 versus time (Days).

FIG. 3 is a plot of the amount of hGH in serum (ng/mL) at predeterminedintervals over a 24 day period following administration ofhGH-containing microparticle formulations described herein versus time(Days).

DETAILED DESCRIPTION OF THE INVENTION

The features and other details of the composition and method of theinvention, will now be more particularly described with references tothe accompanying drawings and pointed out in the claims. It isunderstood that the particular embodiments of the invention are shown byway of illustration and not as limitations of the invention. Theprinciple features of this invention can be employed in variousembodiments without departing from the scope of the invention.

“Particle size” as that term is used herein refers to a volume medianparticle size as measured by conventional particle size measuringtechniques well known to those skilled in the art, such as, laserdiffraction, photon correlation spectroscopy, sedimentation field flowfractionation, disk centrifugation or electrical sensing zone. Laserdiffraction is preferred.

As used herein, the term “micron particles” refers to particles having avolume median particle size of less than about 2 microns. In a preferredembodiment, the micron particles are submicron particles.

As used herein, the term “submicron particles” refers to particleshaving a volume median particle size of less than 1 micron. The volumemedian particle size is the median diameter of the volume-weighted sizedistribution.

The method of the invention for preparing a composition for thesustained release of a labile agent, comprises the steps of:

a) forming a suspension comprising the labile agent dispersed in apolymer solution comprising at least one biocompatible polymer and atleast one polymer solvent;

b) wet milling the suspension to achieve micron particles of the labileagent; and

c) removing the polymer solvent thereby forming a solid polymer/labileagent matrix.

The method can further comprise the step of forming droplets of themilled suspension prior to removal of the polymer solvent. Further, themethod can comprise freezing the droplets prior to removal of thepolymer solvent. According to the method of the invention, the dropletscan be microdroplets. In a specific embodiment wherein droplets areformed and then frozen, the polymer solvent can be removed by anevaporation and/or extraction process. In a preferred embodiment, themicron particles are submicron in size.

The composition for sustained release of a labile agent is likewiseprepared according to the method of the invention. In other words, thecomposition for the sustained release of a labile agent as describedherein is a composition prepared by the method comprising the steps of:

a) forming a suspension comprising the labile agent dispersed in apolymer solution comprising at least one biocompatible polymer and atleast one polymer solvent;

b) wet milling the suspension to achieve micron particles of the labileagent; and

c) removing the polymer solvent thereby forming a solid polymer/labileagent matrix.

The method for preparing micron particles of a labile agent comprisesthe step of (a) forming a suspension comprising the labile agent,dispersed in a polymer solution comprising at least one biocompatiblepolymer and at least one polymer solvent; and (b) wet milling thesuspension.

The micron particles of labile agent, as described herein, are preparedaccording to the method of the invention. Consequently, the micronparticles of labile agent are prepared by forming a suspensioncomprising the labile agent, dispersed in a polymer solution comprisingat least one biocompatible polymer and at least one polymer solvent, andwet milling the suspension.

A “labile agent” as that term is used herein, is a protein, polypeptideor oligonucleotide, or the pharmaceutically acceptable salt thereof,which is in its molecular, biologically active form when released invivo, thereby possessing the desired therapeutic, prophylactic and/ordiagnostic properties in vivo. Suitable proteins include, but are notlimited to, immunoglobulins, antibodies, cytokines (e.g., lymphokines,monokines, chemokines), interleukins, interferons, erythropoietin,nucleases, tumor necrosis factor, colony stimulating factors, insulin,enzymes (e.g., superoxide dismutase, a plasminogen activator), tumorsuppressors, blood proteins, hormones and hormone analogs (e.g., agrowth hormone such as, human growth hormone (hGH), adrenocorticotropichormone, leutinizing hormone releasing hormone (LHRH)), vaccines (e.g.,tumoral, bacterial and viral antigens), antigens, growth factors andblood coagulation factors. Suitable polypeptides include proteininhibitors, protein antagonists, and protein agonists. Examples ofoligonucleotides suitable for use in the invention include, but are notlimited to, antisense molecules, ribozymes, antisense oligonucleotides,peptide nucleic acids, decoy RNAs and “dumbbell” DNAs also known astranscription factor decoy DNAs.

In one embodiment, the labile agent is stabilized. The labile agent canbe stabilized against degradation, loss of potency and/or loss ofbiological activity, all of which can occur during formation of themicron particles, during formation of the sustained release compositionhaving the micron particles dispersed therein, and/or prior to andduring in vivo release of the labile agent. In one embodiment,stabilization can result in a decrease in the solubility of the labileagent, the consequence of which is a reduction in the initial release oflabile agent, in particular, when release is from a sustained releasecomposition. In addition, the period of release of the labile agent canbe prolonged.

Stabilization of the labile agent can be accomplished, for example, bythe use of a stabilizing agent. “Stabilizing agent”, as that term isused herein, is any agent which binds or interacts in a covalent ornon-covalent manner or is included with the labile agent. Stabilizingagents suitable for use in the invention are described in co-pendingU.S. patent application Ser. No. 08/934,830 to Burke et al., filed onSep. 22, 1997 and U.S. Pat. No. 5,711,968 to Tracy et al., U.S. Pat.Nos. 5,654,010 and 5,667,808 to Johnson et al., and U.S. Pat. Nos.5,716,644 and 5,674,534 to Zale et al., the entire teachings of whichare incorporated herein by reference. For example, a metal cation can becomplexed with the labile agent, or the labile agent can be complexedwith a polycationic complexing agent such as protamine, albumin,spermidine and spermine, or associated with a “salting-out” salt.

Suitable metal cations include any metal cation capable of complexingwith the labile agent. A metal cation-stabilized labile agent, asdefined herein, comprises a labile agent and at least one type of metalcation wherein the cation is not significantly oxidizing to the labileagent. In a particular embodiment, the metal cation is multivalent, forexample, having a valency of +2 or more. It is preferred that the metalcation be complexed to the labile agent.

Suitable stabilizing metal cations include biocompatible metal cations.A metal cation is biocompatible if the cation is non-toxic to therecipient, in the quantities used, and also presents no significantdeleterious or untoward effects on the recipient's body, such as asignificant immunological reaction at the injection site. Thesuitability of metal cations for stabilizing labile agents and the ratioof metal cation to labile agent needed can be determined by one ofordinary skill in the art by performing a variety of stabilityindicating techniques such as polyacrylamide gel electrophoresis,isoelectric focusing, and HPLC analyses (e.g., Size Exclusion, ReversedPhase and other Ion Exchange) on particles of metal cation-stabilizedlabile agents prior to and following particle size reduction and/orencapsulation. The molar ratio of metal cation to labile agent istypically between about 1:2 and about 100:1, preferably between about2:1 and about 10:1.

Examples of stabilizing metal cations include, but are not limited to,K⁺, Zn⁺², Mg⁺² and Ca⁺². Stabilizing metal cations also include cationsof transition metals, such as Cu⁺². Combinations of metal cations canalso be employed. In a particular embodiment, Zn⁺² is used as astabilizing metal cation for hGH at a zinc cation component to hGH molarratio of about 4:1 to about 100:1. In a preferred embodiment, the zinccation component to hGH molar ratio is about 4:1 to about 10:1, and mostpreferably 10:1.

The labile agent can also be stabilized with at least one polycationiccomplexing agent. Suitable polycationic complexing agents include, butare not limited to, protamine, and albumin. The suitability ofpolycationic complexing agents for stabilizing labile agents can bedetermined by one of ordinary skill in the art in the manner describedabove for stabilization with a metal cation. An equal weight ratio ofpolycationic complexing agent to labile agent is suitable.

The suspension which is milled comprises a labile agent dispersed in apolymer solution. The concentration of the labile agent in thesuspension can be in the range from between about 0.01% to about 50%w/w, preferably from between about 0.1% to about 30% (w/w) of thecombined weight of the polymer and labile agent.

The use of a polymer solution allows particle size reduction of thelabile agent to occur without significant degradation and aggregation ofthe labile agent. The polymer solution comprises a biocompatible polymerwhich is solubilized in a suitable polymer solvent at a concentrationwhich results in achieving milling of the labile agent withoutaggregation or degradation of said agent. A suitable concentration forthe polymer solution can range between about 0.1% w/v to about 100% w/v,more preferably between about 1% w/v to about 30% w/v. The concentrationof the polymer needed to achieve the desired micron particles of labileagent can be determined as described herein. The polymer solution cancomprise one or more solvents.

A suitable polymer solvent is a solvent in which the polymer is solublebut in which the labile agent is substantially insoluble andnon-reactive resulting in suspension of the labile agent in the polymersolution. Examples of suitable polymer solvents include organic liquids,such as methylene chloride, chloroform, ethyl acetate,dimethylsulfoxide, methyl acetate, hexafluoroisopropanol, acetone andcombinations thereof

A polymer is biocompatible if the polymer and any degradation productsof the polymer are non-toxic to the recipient and also possess nosignificant deleterious or untoward effects on the recipient's body,such as a significant chronic immunological reaction at the injectionsite.

“Biodegradable”, as defined herein, means the composition will degradeor erode in vivo to form smaller chemical species. Degradation canresult, for example, by enzymatic, chemical and/or physical processes.Suitable biocompatible, biodegradable polymers include, for example,poly(lactide)s, poly(glycolide)s, poly(lactide-co-glycolide)s,poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolicacid)s, poly(caprolactone), polycarbonates, polyesteramides,polyanhydrides, poly(amino acid)s, poly(ortho ester)s, polyacetals,polycyanoacrylates, polyamides, polyacetals, poly(ether ester)s,copolymers of poly(ethylene glycol) and poly(ortho ester)s,poly(dioxanone)s, poly(alkylene alkylate)s, biodegradable polyurethanes,blends or copolymers thereof.

Biocompatible, non-biodegradable polymers suitable for use in theinvention, include, for example, polyacrylates, polymers ofethylene-vinyl acetates and acyl substituted cellulose acetates,non-degradable polyurethanes, polystyrenes, polyvinyl chloride,polyvinyl fluoride, poly (vinyl imidazole), chlorosulphonatepolyolefins, polyethylene oxide, blends and copolymers thereof.

Further, the terminal functionalities or pendant groups of the polymerscan be modified, for example, to modify hydrophobicity, hydrophilicityand/or provide, remove or block moieties which can interact with theactive agent (via, for example ionic or hydrogen bonding).

Acceptable molecular weights for polymers used in this invention can bedetermined by a person of ordinary skill in the art taking intoconsideration factors such as the desired polymer degradation rate,physical properties such as mechanical strength, and rate of dissolutionof polymer in solvent and viscosity. Typically, an acceptable range ofmolecular weight is about 2,000 Daltons to about 2,000,000 Daltons. In apreferred embodiment, the polymer is a biodegradable polymer orcopolymer. In a more preferred embodiment, the polymer ispoly(lactide-co-glycolide) (herein after “PLG”) with a lactide:glycolideratio of about 1:1 and a molecular weight of about 5,000 Daltons toabout 70,000 Daltons. In an even more preferred embodiment, themolecular weight of the PLG used in the present invention has amolecular weight of about 5,000 Daltons to about 42,000 Daltons.

Wet milling of the suspension comprising the labile agent dispersed in apolymer solution can be accomplished by adding a grinding media to thesuspension and applying a mechanical means to reduce the particle sizeof the labile agent to a volume median particle size of about 2 micronsor less, preferably less than about 1 micron (submicron). The mechanicalmeans applied to reduce the particle size, can take the form of adispersion mill or any blender. Suitable dispersion mills include arotary mill, a ball mill, an attritor mill, a vibratory mill, aplanetary mill, media mills such as a sand mill, and a bead mill. Arotary blender such as the Shaker Mixer, TURBULA® Type T2C, availablefrom Glen Mills of Clifton N.J.) is preferred. When using the TURBULA®Shaker Mixer, the particle size of the sample is reduced by rotating acontainer having the sample and grinding media disposed therein.

The grinding media can be selected from rigid media preferably sphericalor particulate in form having an average size less than about 5 mm,preferably less than about 3 mm. In selecting a material for use as agrinding media the material should be harder than the labile agent andstable in the milling medium to achieve successful milling. Examples ofmedia suitable for use in the invention include zirconium silicate,zirconium oxide, such as 95% ZrO stabilized with magnesia, glass,stainless steel, titania, alumina, and 95% ZrO stabilized with yttrium.

The milling time can vary and can depend upon, for example, thecomposition of the suspension being milled, the grinding media employed,and the mechanical means being applied. For example, the particle sizeof the labile agent at the commencement of milling is an importantfactor in the length of time needed to achieve micron particles oflabile agent. Advantageously, the particle size of the suspension can bedetermined at any time during the milling process by removing a sampleof the suspension and performing particle size analysis using thetechniques described above. Typically, the suspension is milled forbetween about 24 and about 72 hours.

Milling of the suspension should be conducted at a temperature whichdoes not significantly degrade the labile agent. As such, a suitablemilling temperature can be determined based on the labile agent present.Typically, the milling temperature will be less than about 30° C. In apreferred embodiment, milling is conducted at about 10° C. In a morepreferred embodiment milling is conducted at about 4° C.

The term “sustained release composition” as defined herein, comprises apolymer and micron particles of a labile agent (also referred to hereinas a “polymer/labile agent matrix”). The polymers of the invention arebiocompatible. Suitable biocompatible polymers, can be eitherbiodegradable or non-biodegradable polymers or blends or copolymersthereof, as described herein.

The sustained release compositions of this invention can be formed intomany shapes such as a film, a pellet, a cylinder, a disc or amicroparticle. A “microparticle” as defined herein, comprises a polymercomponent having a diameter of less than about one millimeter and havingmicron particles of labile agent dispersed therein. A microparticle canhave a spherical, non-spherical or irregular shape. Typically, themicroparticle will be of a size suitable for injection. A preferred sizerange for microparticles is from about one to about 180 microns indiameter.

As defined herein, a sustained release of labile agent is release of thelabile agent from a biocompatible polymer matrix which occurs over aperiod which is longer than that period during which a biologicallysignificant amount of the labile agent, would be available followingdirect administration of a solution of the labile agent. It is preferredthat a sustained release be a release of labile agent which occurs overa period of greater than two days. A sustained release of labile agent,from a polymer matrix can be a continuous or a discontinuous release,with relatively constant or varying rates of release. The continuity ofrelease and level of release can be affected by the type of polymercomposition used (e.g., monomer ratios, molecular weight, and varyingcombinations of polymers), labile agent loading, and/or selection ofexcipients to produce the desired effect.

The amount of stabilized labile agent, which is contained within thepolymer/labile agent matrix of a sustained release composition, is atherapeutically effective amount which can be determined by a person ofordinary skill in the art, taking into consideration factors such asbody weight, condition to be treated, type of polymer used, and releaserate from the polymer. A “therapeutically effective amount”, as usedherein, is the amount of the composition for the sustained release of alabile agent from a polymer matrix, necessary to elicit the desiredbiological response following administration.

Typically, the sustained release composition can contain from about0.01% (w/w) to about 50% (w/w) of labile agent (dry weight ofcomposition). The amount of agent used will vary depending upon thedesired effect of the agent, the planned release levels, and the timespan over which the agent will be released. A preferred range of agentloading is between about 0.1% (w/w) to about 30% (w/w) agent. A morepreferred range of agent loading is between about 0.5% (w/w) to about20% (w/w) agent.

In another embodiment, the sustained release composition can containexcipients. The excipients can be added to the suspension prior to orfollowing milling. These excipients are added to maintain the potency ofthe labile agent over the duration of release and modify polymerdegradation. Suitable excipients include, for example, carbohydrates,amino acids, fatty acids, surfactants, and bulking agents, and are knownto those skilled in the art. The amount of excipient used is based onratio to the labile agent, on a weight basis. For amino acids, fattyacids and carbohydrates, such as sucrose, lactose, mannitol, dextran andheparin, the ratio of carbohydrate to labile agent, is typically betweenabout 1:10 and about 20:1. For surfactants, such as TWEEN™ andPLURONIC™, the ratio of surfactant to labile agent is typically betweenabout 1:1000 and about 1:20.

Bulking agents typically comprise inert materials. Suitable bulkingagents are known to those skilled in the art.

The excipient can also be a metal cation component which is separatelydispersed within the polymer matrix. This metal cation component acts tomodulate the release of the labile agent, by for example, modifyingpolymer degradation and is not complexed with the labile agent. Themetal cation component can optionally contain the same species of metalcation, as is contained in the metal cation stabilized labile agent,and/or can contain one or more different species of metal cation. Themetal cation component acts to modulate the release of the labile agentfrom the polymer matrix of the sustained release composition and canenhance the stability of the labile agent in the composition. A metalcation component used in modulating release typically comprises at leastone type of multivalent metal cation. Examples of metal cationcomponents suitable to modulate release include or contain, for example,Mg(OH)₂, MgCO₃ (such as 4MgCO₃.Mg(OH)₂.5H₂O), MgSO₄, Zn(OAc)₂, Mg(OAc)₂,ZnCO₃ (such as 3Zn(OH)₂2ZnCO₃), ZnSO₄, ZnCl₂, MgCl₂, CaCO₃, zinc citrateand magnesium citrate. A suitable ratio of metal cation component topolymer is between about 1:99 to about 1:2 by weight. The optimum ratiodepends upon the polymer and the metal cation component utilized. Apolymer matrix containing a dispersed metal cation component to modulatethe release of a biologically active agent from the polymer matrix isfurther described in U.S. Pat. No. 5,656,297 to Bernstein et al. andco-pending U.S. patent application Ser. No. 09/056,566 filed on Apr. 7,1998, the teachings of both of which are incorporated herein byreference in their entirety.

In yet another embodiment, at least one pore forming agent, such as awater soluble salt, sugar or amino acid, is included in the sustainedrelease composition to modify the microstructure. The proportion of poreforming agent added to the suspension comprising micron particles oflabile agent dispersed in a solution comprising at least onebiocompatible polymer and at least one polymer solvent, is between about1% (w/w) to about 30% (w/w). It is preferred that at least one poreforming agent be included in a nonbiodegradable polymer matrix of thepresent invention.

Suitable methods for forming a composition for the sustained release oflabile agent are described in U.S. Pat. No. 5,019,400, issued to Gombotzet al., and co-pending U.S. patent application Ser. No. 08/443,726,filed May 18, 1995, the teachings of which are incorporated herein byreference in their entirety. This method of formation, as compared withother methods such as phase separation, can also reduce the amount oflabile agent required to produce a sustained release composition with aspecific labile agent content.

In this method, a suspension comprising micron particles of the labileagent dispersed in a solution comprising at least one biocompatiblepolymer and at least one polymer solvent is processed to createdroplets, wherein at least a significant portion of the dropletscontains polymer, polymer solvent and the micron particles of labileagent. These droplets are then frozen by a suitable means. Examples ofmeans for processing the suspension to form droplets include directingthe dispersion through an ultrasonic nozzle, pressure nozzle, Rayleighjet, or by other known means for creating droplets from a solution.

Means suitable for freezing droplets include directing the droplets intoor near a liquified gas, such as liquid argon or liquid nitrogen to formfrozen microdroplets which are then separated from the liquid gas. Thefrozen microdroplets are then exposed to a liquid or solid non-solvent,such as ethanol, hexane, ethanol mixed with hexane, heptane, ethanolmixed with heptane, pentane or oil.

The solvent in the frozen microdroplets is extracted as a solid and/orliquid into the non-solvent to form a polymer/labile agent matrixcomprising a biocompatible polymer and micron particles of a labileagent. Mixing ethanol with other non-solvents, such as hexane, heptaneor pentane, can increase the rate of solvent extraction, above thatachieved by ethanol alone, from certain polymers, such aspoly(lactide-co-glycolide) polymers.

A wide range of sizes of sustained release compositions can be made byvarying the droplet size, for example, by changing the ultrasonic nozzlediameter. If the sustained release composition is in the form ofmicroparticles, and very large microparticles are desired, themicroparticles can be extruded, for example, through a syringe directlyinto the cold liquid. Increasing the viscosity of the polymer solutioncan also increase microparticle size. The size of the microparticleswhich can be produced by this process ranges, for example, from greaterthan about 1000 to about 1 micrometers in diameter.

Yet another method of forming a sustained release composition, from asuspension comprising a biocompatible polymer and micron particles of alabile agent, includes film casting, such as in a mold, to form a filmor a shape. For instance, after putting the suspension into a mold, thepolymer solvent is then removed by means known in the art, or thetemperature of the polymer suspension is reduced, until a film or shape,with a consistent dry weight, is obtained. Film casting of a polymersolution, is further described in U.S. Pat. No. 5,656,297, the teachingsof which are incorporated herein by reference in their entirety.

Without being bound by a particular theory it is believed that therelease of the labile agent can occur by two different mechanisms.First, the labile agent can be released by diffusion through aqueousfilled channels generated in the polymer matrix, such as by thedissolution of the labile agent, or by voids created by the removal ofthe polymer solvent during the preparation of the sustained releasecomposition. A second mechanism is the release of the labile agent, dueto degradation of the polymer. The rate of degradation can be controlledby changing polymer properties that influence the rate of hydration ofthe polymer. These properties include, for instance, the ratio ofdifferent monomers, such as lactide and glycolide, comprising a polymer;the use of the L-isomer of a monomer instead of a racemic mixture; andthe molecular weight of the polymer. These properties can affecthydrophilicity and crystallinity, which control the rate of hydration ofthe polymer. Hydrophilic excipients such as salts, carbohydrates, andsurfactants can also be incorporated to increase hydration which canalter the rate of erosion of the polymer.

By altering the properties of the polymer, the contributions ofdiffusion and/or polymer degradation to labile agent release can becontrolled. For example, increasing the glycolide content of apoly(lactide-co-glycolide) polymer and decreasing the molecular weightof the polymer can enhance the hydrolysis of the polymer, and thusprovides an increased labile agent release from polymer erosion.

In addition, the rate of polymer hydrolysis is increased in non-neutralpH. Therefore, an acidic or a basic excipient can be added to thepolymer suspension, used to form the sustained release composition, forexample, microparticles, to alter the polymer erosion rate.

The composition of this invention can be administered to a human, orother animal, by injection, implantation (e.g., subcutaneously,intramuscularly, intraperitoneally, intracranially, and intradermally),administration to mucosal membranes (e.g., intranasally, intravaginally,intrapulmonary or by means of a suppository), or in situ delivery (e.g.,by enema or aerosol spray) to provide the desired dosage of labile agentbased on the known parameters for treatment with the particular agent ofthe various medical conditions.

Exemplifications

Polymer

The polymers employed in the following examples are described below:

RG 502H: 50:50 poly(D,L-lactide-co-glycolide) (PLG) with hydrophilic endgroups, nominal MW 10 k Daltons purchased from Boehringer IngelheimChemicals, Inc. of Montvale, N.J.

MEDISORB® 5050 2A: 50:50 poly(D,L-lactide-co-glycolide) (PLG) withhydrophilic end groups, nominal MW 8 k Daltons, purchased from Alkermes,Inc. of Cincinnati, Ohio.

General Process for the Preparation of Polymer Microparticles

Forming droplets of the milled suspension by atomizing the milledsuspension comprising submicron particles of at least one labile agentdispersed in a solution of at least one biocompatible polymer, at leastone polymer solvent and any excipients.

Freezing the droplets by contact with liquid nitrogen.

Extracting the polymer solvent from the frozen droplets into anextraction solvent (e.g., ⁻80° C. ethanol), thereby formingmicroparticles comprising a polymer/labile agent matrix.

Isolating the microparticles from the extraction solvent by filtration.

Removing any remaining solvent from the microparticles.

Sizing of the microparticles by passage through an appropriately sizedmesh.

EXAMPLE 1 Preparation of Submicron Particles of Zinc-Complexed hGH

Step A. Formation of Zn⁺²:hGH Protein Complex

Bulk hGH whose DNA sequence is described in U.S. Pat. No. 4,898,830,issued to Goeddel et al. was provided at 20 mg/mL in 25 mM sodiumbicarbonate. It was complexed with 54.5 mM zinc acetate at a 10:1 zincto hGH ratio.

The suspension of Zn⁺²-complexed hGH was atomized using an ultrasonicnozzle (Type V1A: Sonics and Material, Danbury, Conn.) and sprayed intoa polypropylene tub containing liquid nitrogen to form frozen particles.The polypropylene tub was then placed into a ⁻80° C. freezer until theliquid nitrogen evaporated. The frozen particles, which containedZn⁺²-complexed hGH, were then lyophilized to yield dry Zn:hGH powder.

Step B. Milling of Particles

400 mg of zinc-complexed hGH, prepared according to Step A, wassuspended in 15 mL of a 10% solution of PLG (RG 502H) in methylenechloride. The suspension was milled at approximately 4° C. using arotary blender (TURBULA® Model T2C available from Glen Mills of Clifton,N.J.) and 10 g of 100 μm zirconia silicate beads. Milling time wasapproximately 48 hours.

Particle size analysis was conducted on the suspension upon thecompletion of milling using a Coulter LS 130 laser diffractioninstrument with small volume nodule. The particles of the milledsuspension had a volume median particle size of 0.2 microns. Particlesize analysis was also conducted on a sample of the Zn⁺²-complexed hGHprior to milling, and on a sample of Zn⁺²-complexed hGH which had beensonicated for approximately four minutes with a tapered microtip in anice bath, as an alternative method of particle size reduction. Resultsshowed that the unprocessed complex had a volume median particle size of16.2 μm and the sonicated complex had a volume median particle size of 3μm.

EXAMPLE 2 Encapsulation and in Vitro Release of hGH

Two separate formulations of microparticles containing the milled andsonicated Zn⁺²-complexed hGH particles described in Step B of Example 1,were prepared using the “General Process” described above. In each case,the differently sized particles were present in their respectivesolutions (10% RG 502H in methylene chloride) at an amount necessary toachieve a theoretical load of 16% in the final microparticle. ZnCO₃,sufficient for a 1% load in the final microparticle, was also present atthe onset of the “General Process.”

The two microparticle formulations formed as described above, and havingmilled and sonicated Zn⁺²-complexed hGH incorporated therein, wereevaluated for their in vitro release of drug over the first 24 hours.The hGH-containing microparticles were hydrated in buffer (150 mMphosphate with 0.1% Pluronic™ F68 at pH 7.3) at 4° C. A sample of bufferwas removed following the first 24 hours of incubation. Released proteinwas quantified by BioRad Protein Assay (BioRad, Inc., Richmond, Calif.).The initial release of hGH from the microparticles containing thesonicated protein (volume median particle size 3.3 μm) over the first 24hours was approximately 48%. The initial release of hGH from themicroparticles containing the milled protein (volume median particlesize 0.2 μm) over the first 24 hours was 6%.

The results show that hGH-containing microparticles, wherein theparticle size of the incorporated hGH is in the submicron range, exhibita reduction in the initial release of protein when compared tohGH-containing microparticles wherein the particle size of theincorporated protein has a volume median particle size of 3.3 microns.

EXAMPLE 3

Five solutions, each containing 250 mgs of Zn⁺²-complexed hGH preparedaccording to Example 1, 1 g of MEDISORB® 5050 2A polymer, and 17 mL ofmethylene chloride were prepared. All solutions were sonicated for fourminutes prior to milling using a microtip probe available from Sonicsand Material, Danbury Conn. 10 gms of zirconium silicate beads (100 μmdiameter) were added to each solution. Four of the solutions were wetmilled for 4.5, 22, 48, and 72 hours, and are identified as solutions2-5, respectively of Table 1. Solution 1 was not subjected to themilling process. Milling was conducted at a temperature of about 4 ° C.,using a TURBULA® blender Model T2C at a speed of approximately 96 r.p.m.

At each end point, a sample of solution was removed and subjected toparticle size analysis using a Coulter LS 130. The volume medianparticle size of solutions 1-5 is presented in Table 1.

TABLE 1 Solution Milling Time Volume Median Particle Size ofIdentification (Hours) Zn⁺²-complexed hGh (Microns) 1 0 3.619 2 4.51.244 3 22 0.590 4 48 0.436 5 72 0.416

EXAMPLE 4 Encapsulation and in Vitro Release hGH

Part A: Microparticle Formation

Microparticles containing the Zn⁺²-complexed hGH particles of Solutions1-5 of Example 4 and 1% ZnCO₃ were prepared using the “General Process”described above. A description of each microparticle formulationprepared is presented in Table 2. The number designation of theformulations in Table 2 reflects encapsulation of the solution havingthe same number designation as in Table 1 of Example 3.

Part B: In Vitro Release

Microparticle Formulations 1-5 were evaluated for their in vitro releaseof drug over the first 24 hours. Duplicate microparticle formulationswere hydrated in buffer (50 mM HEPES, 10 mM KCl, 0.1% NaN₃) at 37° C.The amount of protein released at 24 hours post hydration was quantifiedusing the BioRad Protein Assay (BioRad, Inc. Richmond, Calif.). Theresults of the assay are presented in Table 2.

TABLE 2 Amount of Amount of Micro- Micro- Actual hGH in % hGH ParticleParticles hGH Micro- Released Formulation Assayed Load Particles OverFirst Average Identification (mgs) (% w/w) (μgs) 24 Hours (SD) 1 9.517.4 1653 28 29.3 8.3 17.4 1444.2 30 (1.3%) 2 8.2 16.8 1377.6 15 15.48.0 16.8 1344 15 (0.2%) 3 9.5 16.9 1605.5 8 8.3 8.8 16.9 1487.2 9 (0.4%)4 9.1 16.8 1528.8 5 4.8 8.8 16.8 1478.4 5 (0.3%) 5 9.3 16.7 1553.1 4 4.09.8 16.7 1636.6 4 (0.4%)

The in vitro release as determined above for Formulations 1-5 is plottedas a function of the volume median particle size in FIG. 1. The resultsshow that release over the first 24 hours is directly related to thevolume median particle size of the protein prior to encapsulation.

EXAMPLE 5 In Vivo Release hGH

Studies were conducted in rats to determine the in vivo release rate ofhGH from Microparticle Formulations 1-5 of Example 4 over the first 24hours following treatment. Male Sprague-Dawley rats were injectedsubcutaneously with a dose of approximately 15 mg/kg of hGH. The amountof Formulations 1-5 needed to achieve this dose was based on the %theoretical load of the formulations. The microparticles were suspendedin 0.75 mL of an aqueous injection vehicle comprising 3% CMC (lowviscosity), 0.1% polysorbate 20, in 0.9% NaCl.

Blood samples were collected at the following intervals: 0.08, 0.17,0.25, 0.42 and 1.00 days. The blood samples were clotted and hGHconcentrations in serum were determined using an ELISA provided in arhGH kit available from Boehringer Mannheim (Catalog No.: 15868). Theresults are presented in Table 3 as an average of the three test animalsin each treatment group.

TABLE 3 Microparticle Timepoint Average Concentration Formulation (Days)of hGH in Serum (ng/mL) 1 0.08 1933.67 0.17 1642.33 0.25 1701.00 0.421275.00 1.00 41.87 2 0.08 838.33 0.17 690.00 0.25 907.33 0.42 460.331.00 62.40 3 0.08 392.00 0.17 405.67 0.25 442.50 0.42 274.67 1.00 24.234 0.08 228.00 0.17 229.33 0.25 216.40 0.42 148.00 1.00 40.40 5 0.08211.67 0.17 269.33 0.25 302.50 0.42 171.45 1.00 18.37

The results presented in Table 3 demonstrate that the in vivo releasefrom Microparticle Formulations 1-5 over the first 24 hours followingadministration, decreases as the particle size of the Zn⁺²-complexed hGHdecreases. These results are presented graphically in FIG. 2. Theaverage Cmax (ng/mL) for each treatment group over the first 24 hourperiod following administration is plotted as a function of particlesize of the Zn⁺²-complexed hGH prior to encapsulation in FIG. 1. Theplot shows that C_(max) is directly related to the size of theZn⁺²-complexed hGH prior to encapsulation.

EXAMPLE 6 In Vivo Release of hGH

Microparticles containing 16% hGH, 1% ZnCO₃ and MEDISORB® 5050 2Apolymer were prepared according to the procedures outlined above for thepreparation of Microparticle Formulation 5. The volume median particlesize of the Zn⁺²-complexed hGH prior to encapsulation was determined tobe 0.5 microns.

The release of hGH from microparticles having the 0.5 micron labileagent incorporated therein and Microparticle Formulation 1 (Control)were evaluated in vivo according to the method described in Example 5,but over a 24 day period.

The hGH concentration in serum (ng/mL) was plotted as a function of timein FIG. 3. The plot shows a decrease in the C_(max) and a longer periodof sustained release for the microparticles having Zn⁺²-complexed hGHwith a particle size of 0.5 microns as compared to the control.

EXAMPLE 7 Wet Milling with Pluronic™ F68 and/or PLG

Particles of Zn⁺²-complexed hGH were prepared using a 10:1 molar ratioof zinc component to hGH according to the method of Example 1. TheZn⁺²-complexed hGH was separately suspended in each of the followingsolutions to achieve a final concentration of 22 mg/mL of hGH inmethylene chloride:

A—1% solution of F68 in methylene chloride.

B—10% solution of poly(lactic-co-glycolic acid) (PLG RG502H) inmethylene chloride; and

C—10% solution of poly(lactic-co-glycolic acid) (PLG RG502H) inmethylene chloride with 1% F68.

Suspensions A, B and C were wet milled for 48 hours at a temperature ofapproximately 4° C. Particle size analysis was performed using a CoulterLS130. The results are presented below.

Suspension A:

Approximately 48% of the total volume was made up of particles smallerthan a micron, with the remainder made up of particles between 1 and 10μm. The volume median particle size in this case was 1.2 μm.

Suspensions B and C:

Approximately 95% of the total volume consisted of particles smallerthan 1 μm, with a volume median particle size of 210 nm.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the spirit and scope of theinvention as defined by the appended claims. Those skilled in the artwill recognize or be able to ascertain using no more than routineexperimentation, many equivalents to the specific embodiments of theinvention described specifically herein. Such equivalents are intendedto be encompassed in the scope of the claims.

What is claimed is:
 1. A method for preparing a composition for the sustained release of a labile agent, comprising the steps of: a) forming a suspension comprising the labile agent dispersed in a polymer solution comprising at least one biocompatible polymer and at least one polymer solvent; b) wet milling the suspension to achieve submicron particles of the labile agent; and c) removing the polymer solvent thereby forming a solid polymer matrix having the labile agent dispersed therein.
 2. The method of claim 1 wherein the submicron particles have a volume median particle size of less than 1 micron, measured by laser diffraction.
 3. The method of claim 1 wherein step (b) is conducted at a temperature of less than about 30° C.
 4. The method of claim 3 wherein the temperature is less than about 10° C.
 5. The method of claim 3 wherein the temperature is less than about 4° C.
 6. The method of claim 1 wherein the labile agent is present in the suspension at a concentration of from about 0.01 to about 50% w/w of the combined weight of polymer and labile agent.
 7. The method of claim 6 wherein the labile agent is present at a concentration of about 0.01 to 30% w/w of the combined weight of the polymer and labile agent.
 8. The method of claim 1 wherein the labile agent is a protein.
 9. The method of claim 1 wherein the labile agent is complexed to a stabilizing metal cation.
 10. The method of claim 9 wherein said stabilizing metal cation is selected from the group consisting of Zn⁺², Ca⁺², Cu⁺², Mg⁺², K⁺ and any combination thereof.
 11. The method of claim 10 wherein said stabilizing metal cation is Zn⁺².
 12. The method of claim 1 wherein the biocompatible polymer is biodegradable.
 13. The method of claim 12 wherein the biodegradable polymer is selected from the group consisting of poly(lactide)s, poly(glycolide)s, poly(lactide-coglycolide)s, poly(lactic acid)s, poly(glycolic acid)s, poly(lactic acid-co-glycolic acid)s, poly(caprolactone), polycarbonates, polyesteramides, polyanhydrides, poly(amino acid)s, poly(ortho ester)s, polycyanoacrylates, polyamides, polyacetals, poly(ether ester)s, copolymers of poly(ethylene glycol) and poly(ortho ester)s, poly(dioxanone)s, poly(alkylene alkylate)s, biodegradable polyurethanes, blends and copolymers thereof.
 14. The method of claim 12 wherein the biocompatible polymer is non-biodegradable.
 15. The method of claim 1 wherein the polymer solvent is methylene chloride.
 16. The method of claim 1, wherein wet milling comprises the steps of: a) adding a grinding media to the suspension; and b) applying a mechanical means thereto.
 17. The method of claim 16, wherein the mechanical means is a dispersion mill or rotary blender.
 18. The method of claim 17, wherein the dispersion mill is selected from the group consisting of: a rotary mill, a ball mill, an attrition mill, a vibratory mill, a planetary mill, a media mill or a bead mill. 